Abstract The Specific Aim of this Phase I STTR proposal is to test the feasibility of blocking MCJ (also called DnaJC15) to overcome acute acetaminophen liver toxicity. Acetaminophen or APAP (from acetyl-para- aminophenol) is a common drug used to address pain and fever. In the US alone, about 30,000 patients per year are admitted to intensive care units with APAP-induced liver injury and close to 10,000 of these patients undergo liver transplantation. For overdosing, treatment with the antioxidant N-acetylcysteine (NAC) is the only known effective approach. NAC is very effective at reducing APAP-induced liver injury if given within 8 hours after ingestion. However, after about 8 hours, NAC has a minimal chance of rescuing the liver; therefore alternative therapeutic approaches are needed. While the mechanisms underlying APAP-induced acute liver injury are not fully understood, increased oxidative stress and reduction in ATP production due to the effect of APAP on mitochondrial electron transport chain (ETC) seem to play a major role in hepatocyte cell death. Thus, increasing mitochondrial respiration without increasing oxidative stress could be a potential therapeutic approach to address APAP liver toxicity. However, this approach has not been previously tested because there are no available strategies to enhance mitochondrial respiration. MCJ is an endogenous negative regulator of Complex I, a key component of the mitochondrial respiratory chain. The absence of MCJ has been shown to enhance mitochondrial ATP production, without increasing production of reactive oxygen species (ROS). Thus, reducing MCJ could be an approach to protect mitochondria in liver from APAP-induced liver injury. Under physiological conditions, complete removal of MCJ has no obvious toxic effects. Thus, we predicted that blocking MCJ expression could be a relatively safe approach to protect from APAP-induced liver injury. In fact, we have found that siMCJ treatment shows a superior efficacy to NAC in treating APAP-induced liver injury in a mouse model. We have demonstrated highly effective therapeutic efficacy, even when administered 24 hours after APAP administration. To achieve our Specific Aim, we will carry out 4 Tasks: Task 1. Establish the maximum tolerated dose (MTD) or a high tolerated dose of siMCJ. Task 2. To establish a dose/effect of siMCJ on APAP-induced liver injury. Task 3. To establish the effect of siMCJ treatment on long-term recovery from APAP-induced liver injury. Task 4. To validate that the therapeutic effect of siMCJ is maintained in the presence of NAC, the current standard of care treatment for APAP-induced liver injury. Test of Feasibility: The therapeutic index (TI) (i.e. MTD/EC90) must be >1 and long-term (8 week) recovery must be complete enough to prevent the need to sacrifice mice. Maximal efficacy must be maintained when siMCJ is co-delivered with NAC.